This research component has the overall goal to develop and improve methods for characterizing large proteins and protein complexes. Protein interactions are crucial for many biological processes, such as cellular switches, signaling mechanisms, enzyme regulation, gene expression and development. Much is known about structures of tight complexes from crystallography and NMR spectroscopy. However, information on weak complexes is rather sparse due to the limitations of current technologies. This is in contrast to the fact that many protein interactions must be weak and transient, for example to rapidly turn signaling pathways on and off, or to recycle cellular proteins. Furthermore, protein complexes represent an underutilized class of targets for design of drugs against diseases. NMR has unique capabilities for defining structures and states of large proteins and protein complexes that cannot be obtained with crystallography. Major improvements of NMR hardware have recently become available that are only beginning to be efficiently utilized. New ideas of spin physics, control theory, sampling strategies, signal processing, or data analysis are being developed, together with new strategies for sample preparation. All this promises major advances of NMR spectroscopy with large proteins and protein complexes. Here we propose to develop new approaches to make optimal use of modern NMR hardware to gain new insights into structures of large proteins and protein complexes. This will require abandoning some of the traditional procedures for data acquisition and require new data processing methods. This grant has spearheaded such approaches in the past, and similar efforts have appeared in several other laboratories. We propose research towards two specific aims: Aim 1. Develop new NMR experiments for characterization of large proteins systems. The experiments proposed employ heavily non-uniform sampling methods, coherence co-evolution procedures, and they are geared towards optimum use of high-field instruments and use optimum control theory for pulse sequence design. Aim 2. Approaches for characterizing protein complexes. This includes new co-expression methods for facilitating NMR studies of complexes, development of new protein tags to improve the solution behavior of complexes, and NMR and computational techniques for defining the arrangement of proteins in tight and in weak complexes.